Cyclic adenylate receptor protein mutant and application thereof in co-utilization of glucose and xylose

Abstract

The invention discloses a cyclic adenylate receptor protein mutant and application of the cyclic adenylate receptor protein mutant to co-utilization of glucose and xylose. The cyclic adenylate receptor protein mutant disclosed by the invention is any one of the following A1)-A3): A1) a protein as shown in SEQ ID No.1; a2) a protein which is obtained by substituting and / or deleting and / or adding one or more amino acid residues to the amino acid sequence except the 141 site of the protein of A1) and has the same function; and A3) a fusion protein obtained by connecting a label to the N end or / and C end of the protein in A1) or A2). After the cyclic adenylate receptor protein mutant is expressed in recombinant Escherichia coli QJL008, glucose and xylose are used as substrates for fermentation, it is found that the xylose consumption of the cyclic adenylate receptor protein mutant is remarkably increased, the requirement for synchronous and efficient utilization of biomass resources including glucose and xylose is met, and the mutant has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and relates to a cyclic adenosine monophosphate receptor protein mutant and application thereof to glucose-xylose co-utilization. Background Art

[0002] Lignocellulose is the most widely available renewable biomass resource in the world. Lignocellulose is the most abundant biomass source for renewable production of fuels and chemicals. According to statistics, the total annual output of lignocellulose accounts for about 50% of all biological resources, equivalent to about 10-50 billion tons. The production of bioenergy and bulk chemicals by fermentation of lignocellulose hydrolysate can partially get rid of dependence on petroleum resources. Glucose and xylose are the main hydrolyzed monosaccharides in lignocellulose, with glucose accounting for 30%-50% of the hydrolyzed sugars; xylose accounts for 5%-20%. Therefore, the effective synergistic utilization of glucose and xylose is of great significance to the production of fuels and chemicals derived from lignocellulosic biomass.

[0003] Carbon catabolite repression (CCR) is also known as the glucose repression effect, which means that the presence of an easily decomposable carbon source (glucose) in the culture medium inhibits the ability of the strain to metabolize other carbon sources such as xylose, maltose, and lactose. This effect is widely present in bacteria and fungi. Glucose and xylose are present in the hydrolysate of lignocellulosic biomass at the same time. The strain preferentially depletes the glucose in the hydrolysate. After that, the strain synthesizes cyclic adenosine monophosphate cAMP and binds to activate the global transcription factor cAMP receptor protein (CRP), thereby further activating the transcription of operons related to xylose metabolism, causing the strain to begin to consume xylose. Therefore, the strain cannot utilize glucose and xylose simultaneously, resulting in low carbon source utilization efficiency and product synthesis efficiency in the lignocellulosic hydrolysate.

[0004] Escherichia coli has a xylose transporter and a pathway for decomposing xylose, but the transcription of related genes is inhibited in the presence of glucose. Currently, there are many strategies to solve this inhibition and achieve the co-utilization of glucose and xylose. For example, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) elements PtsG or PtsI are inactivated to activate the transcription of the xylose decomposition operon; or the transcriptional activators of the xylose decomposition operon, the cAMP-independent CRP mutants Crp* (I112L, T127I, A144T) and XylR* (R121C or P363S), are overexpressed. However, the above strategies also have defects, such as the effect of the cAMP-independent CRP mutant in activating the target operon is inferior to the wild-type CRP after binding to cAMP; and CRP regulates the expression of more than 400 genes in Escherichia coli. CRP mutants may affect the expression of other key genes, resulting in slower growth of the strain. In addition, the efficiency of glucose utilization of the strains transformed by the above strategy is still higher than that of xylose utilization, and the complete synchronous utilization of glucose and xylose cannot be achieved. Summary of the invention

[0005] The problem to be solved by the present invention is how to improve the ability of Escherichia coli (ie, Escherichia coli) to utilize xylose in an environment where glucose exists.

[0006] In order to solve the above technical problems, the present invention first provides a protein, whose name is CRP G141D, which is any one of the following A1)-A3):

[0007] A1) the protein shown in SEQ ID No. 1;

[0008] A2) a protein having the same function as the protein described in A1) with one or more amino acid residues substituted and / or deleted and / or added to the amino acid sequence except for position 141;

[0009] A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in A1) or A2).

[0010] The CRP G141D protein in A2) above is a protein having 75% or more identity with the amino acid sequence of the protein shown in SEQ ID No.1 and having the same function. Identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gapcost and Lambda ratio to 11, 1 and 0.85 (default value) respectively, and searching for a pair of amino acid sequence identities to calculate, and then the identity value (%) can be obtained. The identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0011] The protein of SEQ ID No. 1 in A2) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0012] The coding gene of the protein of SEQ ID No.1 in A2) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence of SEQ ID No.2, and / or performing missense mutation of one or several base pairs, and / or attaching a coding sequence of a tag at its 5′ end and / or 3′ end. The DNA molecule of SEQ ID No.2 encodes the CRP G141D protein of SEQ ID No.1.

[0013] The tag in A3) can be a polypeptide or protein fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag and / or SUMO tag, etc.

[0014] The present invention also provides a biological material related to CRP G141D, wherein the biological material is any one of the following B1) to B4):

[0015] B1) a nucleic acid molecule encoding CRP G141D;

[0016] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0017] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0018] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0019] In the above biological material, the nucleic acid molecule in B1) may be any one of the following b1)-b3):

[0020] b1) the DNA molecule shown in SEQ ID No. 2 in the sequence list;

[0021] b2) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) and encodes CRP G141D;

[0022] b3) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in b1) or b2) and encodes CRP G141D.

[0023] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0024] A person skilled in the art can easily mutate the nucleotide sequence encoding the CRP G141D protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the CRP G141D protein of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the CRPG141D protein and have the function of the CRP G141D protein.

[0025] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No.1. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0026] In the above biological materials, the stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, and washing at 50°C in 2×SSC, 0.1% SDS.

[0027] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0028] In the above biological material, the expression cassette (CRPG141D gene expression cassette) containing a nucleic acid molecule encoding CRP G141D protein described in B2) refers to a DNA capable of expressing CRP G141D protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the CRP G141D gene, but also a terminator for terminating transcription of the CRP G141D gene. Further, the expression cassette may also include an enhancer sequence.

[0029] Existing expression vectors can be used to construct a recombinant vector containing the CRP G141D gene expression cassette.

[0030] In the above biological materials, the vector may be a plasmid, a cosmid, a phage or a virus vector.

[0031] In the above biological materials, the microorganism can be yeast, bacteria, algae or fungi.

[0032] The present invention also provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli contains a gene encoding CRP G141D.

[0033] The above recombinant Escherichia coli does not contain the gene encoding the protein described in SEQ ID No.3.

[0034] The present invention also provides a method for preparing the recombinant Escherichia coli, wherein the method uses Escherichia coli as a recipient bacterium, and the method comprises: introducing the coding gene of CRP G141D into the genome of the recipient bacterium, or replacing the coding gene of the protein described in SEQ ID No. 3 (i.e., the CRP gene) in the recipient bacterium, to obtain the recombinant Escherichia coli.

[0035] The present invention also provides a method for improving the xylose utilization ability of Escherichia coli in the presence of glucose, the method comprising introducing the coding gene of CRP G141D into the genome of the recipient Escherichia coli, or replacing the coding gene of the protein described in SEQ ID No. 3 in the Escherichia coli, so as to improve the xylose utilization ability of the Escherichia coli in the presence of glucose.

[0036] The use of CRP G141D or the biomaterial in improving the xylose utilization ability of Escherichia coli in the presence of glucose also falls within the protection scope of the present invention.

[0037] The use of CRP G141D or the biological material in the co-utilization of glucose and xylose by Escherichia coli also falls within the protection scope of the present invention.

[0038] The use of the recombinant Escherichia coli in the co-utilization of glucose and xylose by Escherichia coli also falls within the protection scope of the present invention.

[0039] The present invention expresses a cAMP-independent mutant CRP G141D in a recombinant Escherichia coli QJL008 to obtain a strain QJL008-CRP*. With glucose and xylose as substrates, the fermentation is carried out for 24 hours, and the glucose and xylose consumption rates are 0.76 g / gDCW·h and 0.81 g / gDCW·h, respectively. The fermentation is carried out for 96 hours, and 43.62 g / L and 44.33 g / L of glucose and xylose are consumed, respectively, and 87.1 g / L of D-lactic acid is synthesized, thereby meeting the demand for synchronous and efficient utilization of biomass resources glucose and xylose, and having a specific and broad application prospect.

[0040] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0041] Collection Instructions

[0042] Bacteria species: Escherichia coli

[0043] Latin name: Escherichia coli

[0044] Strain ID: QJL010

[0045] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0046] Abbreviation of depository institution: CGMCC

[0047] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0048] Deposit date: May 12, 2023

[0049] Registration number of the Collection Center: CGMCC No.27325 DETAILED DESCRIPTION

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0051] The pRedCas9 plasmid in the following examples has been recorded in the literature "Zhu et al. 2017, The CRISPR / Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway Metab. Eng. 43, 37-

[0052] 45)”, the public can obtain the biological material from the applicant, and the biological material can only be used for repeating the experiments of the present invention and cannot be used for other purposes.

[0053] The placZ plasmid in the following example has been recorded in the document "Qiu Huanna et al., Construction of a stringent promoter on the chromosome of Escherichia coli, 2018, Microbiology Bulletin, 45(8): 1693-1704". The public can obtain the biological material from the applicant. The biological material is only used to repeat the experiment of the present invention and cannot be used for other purposes.

[0054] The pV4 plasmid in the following example has been recorded in "Chinese Patent Application CN114058560A Glycine Production Method", and the public can obtain the biological material from the applicant. The biological material is only used to repeat the experiment of the present invention and cannot be used for other purposes.

[0055] The Escherichia coli ATCC 8739 in the following examples can be obtained by the public from the American Type Culture Collection (ATCC) (https: / / www.atcc.org). The biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0056] The Rec1.0 strain in the following embodiment is a strain obtained by restoring the pgi gene and zwf gene that were knocked out of Escherichia coli AE1.0. Escherichia coli Rce1.0 and AE1.0 have been recorded in the document "Zhu, XN; Fan, FY; Qiu, HN; Shao, MY; Li, D.; Yu, Y.; Bi, CH; Zhang, XL, New xylose transporters support the simultaneous consumption of glucose and xylose in Eescherichiacoli. mLife2022, 1(2), 156-170.", which can be obtained by the public from the applicant. These biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0057] The strains used in the present invention are shown in Table 1, the primers used are shown in Table 2, and the plasmids involved are shown in Table 3. Among them, the strain QJL008 is a strain obtained by restoring the knocked-out 6-phosphoglucose isomerase gene pgi and 6-phosphoglucose dehydrogenase gene zwf of Escherichia coli AE1.0, knocking out the methylglyoxal synthase gene mgsA, the pyruvate formate lyase gene pflB, the fumarate reductase gene frd, and the acetaldehyde / ethanol dehydrogenase gene adhE, and metabolically evolving the strain for 20 generations under the condition that glucose is the only carbon source.

[0058] Table 1. Strains used in the present invention

[0059]

[0060]

[0061] Table 2. Primers used in the present invention

[0062]

[0063]

[0064] Table 3. Plasmids used and constructed in the present invention

[0065]

[0066] The following examples were processed using SPSS11.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0067] Example 1. Construction of strain QJL008-CRP*

[0068] 1. Construction of recombinant Escherichia coli QJL001

[0069] Starting from the recombinant Escherichia coli Rec1.0, the CRISPR / Cas9 dual plasmid gene editing system was used to knock out mgsA (the nucleotide sequence is Genbank No. NC_010468.1, updated on May 16, 2023, positions 2883345-2883803) to obtain the recombinant Escherichia coli QJL001. The specific steps are as follows:

[0070] (1) Construction of pV4-del-mgsA plasmid

[0071] The specific steps of the construction process of pV4-del-mgsA plasmid are as follows:

[0072] The first step is to obtain the pV4 plasmid backbone fragment A. Using the pV4 plasmid as a template, primers N20-B-F1 and N20-B-R1 (specific sequences are shown in Table 2) were used for reverse PCR amplification to obtain a PCR product of about 4.1 kb, which is the pV4 plasmid backbone fragment A. This fragment contains cat, P15A and the self-cleavage element lacI-Ptrc-cat-N20-gRNA.

[0073] In the second step, the mgsA-N20-gRNA sequence fragment B was obtained. Using the pV4 plasmid as a template, PCR amplification was performed with primers mgsA-N20-B-F2 and N20-B-R2 (specific sequences are shown in Table 2) to obtain a PCR product of about 400 bp, which is DNA fragment E.

[0074] The third step is to obtain the upstream and downstream homology arm fragments C and D of the mgsA gene. Using the genomic DNA of Escherichia coli ATCC 8739 as a template, PCR amplification was performed with primers mgsA-F1 and mgsA-R1 (specific sequences are shown in Table 2) to obtain the upstream homology arm fragment C, about 480 bp. Similarly, PCR amplification was performed with primers mgsA-F2 and mgsA-R2 (specific sequences are shown in Table 2) to obtain the downstream homology arm fragment D, about 480 bp.

[0075] In the fourth step, the pV4 plasmid backbone fragment A, the mgsA-N20-gRNA sequence fragment B, and the upstream and downstream homologous arm fragments C and D of the mgsA gene were assembled using the Golden Gate technology strategy (sequentially seamlessly connected in the order of A, B, C and D), and chemically transformed into competent cells TransT1 (Beijing Quanshijin Biotechnology Co., Ltd.). The obtained clones were PCR verified using primers P15A-UP and mgsA-R2 (specific sequences are shown in Table 2), and the band size was about 1.2Kb. The plasmid DNA extracted from the positive clones was sent for sequencing analysis to obtain the correct pV4-del-mgsA plasmid.

[0076] (2) The specific steps of mgsA gene knockout are as follows:

[0077] Starting with the Escherichia coli engineered bacteria Rec1.0, plasmids pRedCas9 and pV4-del-mgsA were simultaneously transformed into Rec1.0 electroporation competent cells, coated with kanamycin and chloramphenicol double-resistance plates, and placed at 30°C overnight for culture. Pick a single clone in 2mL LB (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose), 250r / min speed, 30°C overnight to induce homologous recombination and cut DNA that has not undergone recombination. Dilute and apply LB plates (containing kanamycin and chloramphenicol, 2.5% L(+)-arabinose), and culture at 30°C overnight. Pick a single clone for colony PCR verification, primers are mgsA-YZ-up and mgsA-YZ-down (specific sequences are shown in Table 2), and the positive clones are amplified to obtain PCR products of about 960bp in size, and the positive clones are named recombinant Escherichia coli QJL001.

[0078] 2. Construction of recombinant Escherichia coli QJL004

[0079] Starting from the recombinant Escherichia coli QJL001, the CRISPR / Cas9 dual-plasmid gene editing system was used to knock out pflB (the nucleotide sequence is Genbank No. NC_010468.1, updated on May 16, 2023, positions 2956804-2959086) to obtain the recombinant Escherichia coli QJL004.

[0080] (1) Construction of pV4-del-pflB plasmid

[0081] The specific steps of the construction process of pV4-del-pflB plasmid are as follows:

[0082] The first step is to obtain pV4 plasmid backbone fragment A, which is the same as the first step (1) in step 1.

[0083] In the second step, the pflB-N20-gRNA sequence fragment E was obtained. Using the pV4 plasmid as a template, PCR amplification was performed with primers pflB-N20-B-F2 and N20-B-R2 (specific sequences are shown in Table 2) to obtain a PCR product of about 400 bp, which is the DNA fragment H.

[0084] The third step is to obtain the upstream and downstream homology arm fragments F and G of the pflB gene. Using the genomic DNA of Escherichia coli ATCC 8739 as a template, PCR amplification was performed with primers pflB-F1 and pflB-R1 (specific sequences are shown in Table 2) to obtain the upstream homology arm fragment F, about 480 bp. Similarly, PCR amplification was performed with primers pflB-F2 and pflB-R2 (specific sequences are shown in Table 2) to obtain the downstream homology arm fragment G, about 430 bp.

[0085] In the fourth step, the pV4 plasmid backbone fragment A, pflB-N20-gRNA sequence fragment E, and the upstream and downstream homology arm fragments G and F of the pflB gene were assembled using the Golden Gate technology strategy (sequentially seamlessly connected in the order of A, E, G, and F), and chemically transformed into competent cells TransT1 (Beijing Quanshijin Biotechnology Co., Ltd.). The obtained clones were verified by PCR using primers P15A-UP and pflB-R2, and the band size was about 1.3Kb. The plasmid DNA extracted from the positive clones was sent for sequencing analysis to obtain the correct pV4-del-pflB plasmid.

[0086] (2) The specific steps of pflB gene knockout are as follows:

[0087] Starting from the engineered Escherichia coli QJL001, plasmids pRedCas9 and pV4-del-pflB were simultaneously transformed into QJL001 electroporation competent cells, coated with kanamycin and chloramphenicol double-resistance plates, and placed at 30°C overnight for culture. Pick a single clone in 2mL LB (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose), 250r / min speed, 30°C overnight to induce homologous recombination and cut DNA that has not undergone recombination. Dilute and coat LB plates (containing kanamycin and chloramphenicol, 2.5% L(+)-arabinose), and culture at 30°C overnight. Pick a single clone for colony PCR verification, primers are pflB-YZ-up and pflB-YZ-down, and the positive clones are amplified to obtain PCR products of about 880bp in size, and the positive clones are named recombinant Escherichia coli QJL004.

[0088] 3. Construction of recombinant Escherichia coli QJL005

[0089] Starting from the recombinant Escherichia coli QJL004, the CRISPR / Cas9 dual-plasmid gene editing system was used to knock out frdABCD (the frdA nucleotide sequence of which is Genbank No. NC_010468.1, updated on May 16, 2023, 4214758-4216566; the frdB nucleotide sequence of which is Genbank No. NC_010468.1, updated on May 16, 2023, 4216559-4217293; the frdC nucleotide sequence of which is Genba nk number is NC_010468.1, updated on 16 May 2023, positions 4217304-4217699; the frdD nucleotide sequence is Genbank number NC_010468.1, updated on 16 May 2023, positions 4217710-4218069) and adhE (nucleotide sequence is Genbank number NC_010468.1, updated on 16 May 2023, positions 2627307-2629982), and recombinant Escherichia coli QJL005 is obtained.

[0090] (1) Construction of pV4-del-frd plasmid

[0091] The specific steps of the construction process of pV4-del-frd plasmid are as follows:

[0092] The first step is to obtain pV4 plasmid backbone fragment A, which is the same as the first step (1) in step 1.

[0093] The second step is to obtain the frd-N20-gRNA sequence fragment H. Using the pV4 plasmid as a template, PCR amplification was performed with primers frd-N20-B-F2 and N20-B-R2 (specific sequences are shown in Table 2) to obtain a PCR product of about 400 bp, which is DNA fragment H.

[0094] The third step is to obtain the upstream and downstream homology arm fragments I and J of the frd gene. Using the genomic DNA of Escherichia coli ATCC 8739 as a template, PCR amplification was performed with primers frd-F1 and frd-R1 (specific sequences are shown in Table 2) to obtain the upstream homology arm fragment I, about 420 bp. Similarly, PCR amplification was performed with primers frd-F2 and frd-R2 (specific sequences are shown in Table 2) to obtain the downstream homology arm fragment J, about 400 bp.

[0095] In the fourth step, the pV4 plasmid backbone fragment A, frd-N20-gRNA sequence fragment H, and the upstream and downstream homology arm fragments I and J of the frd gene were assembled using the Golden Gate technology strategy (sequentially seamlessly connected in the order of A, H, I and J), and transformed into the competent cell TransT1 (Beijing Quanshijin Biotechnology Co., Ltd.). The obtained clone was verified by PCR using primers P15A-UP and frd-R2, and the band size was about 1.3Kb. The plasmid DNA extracted from the positive clone was sent for sequencing analysis to obtain the correct pV4-del-frd plasmid.

[0096] (2) The specific steps of frd gene knockout are as follows:

[0097] Starting from the Escherichia coli engineered bacteria QJL004, plasmids pRedCas9 and pV4-del-frd were simultaneously transformed into QJL004 electroporation competent cells, coated with kanamycin and chloramphenicol double-resistance plates, and placed at 30°C overnight culture. Pick a single clone in 2mL LB (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose), 250r / min speed, 30°C overnight to induce homologous recombination and cut DNA that has not undergone recombination. Dilute and apply LB plates (containing kanamycin and chloramphenicol, 2.5% L(+)-arabinose), and culture at 30°C overnight. Pick a single clone for colony PCR verification, primers are frd-YZ-up and frd-YZ-down, and the positive clone bacteria amplify PCR products of about 821bp in size, which are Escherichia coli engineered bacteria with knockout of the frd gene, and then knock out adhE on the basis of knockout of frd.

[0098] (3) Construction of pV4-del-adhE plasmid

[0099] The specific steps of the construction process of pV4-del-adhE plasmid are as follows:

[0100] The first step is to obtain pV4 plasmid backbone fragment A, which is the same as the first step (1) in step 1.

[0101] The second step is to obtain adhE-N20-gRNA sequence fragment H. Using the pV4 plasmid as a template, PCR amplification was performed with primers adhE-N20-B-F2 and N20-B-R2 (specific sequences are shown in Table 2) to obtain a PCR product of about 400 bp, which is DNA fragment H.

[0102] The third step is to obtain the upstream and downstream homology arm fragments I and J of the adhE gene. Using the genomic DNA of Escherichia coli ATCC 8739 as a template, PCR amplification was performed with primers adhE-F1 and adhE-R1 (specific sequences are shown in Table 2) to obtain the upstream homology arm fragment I, about 430 bp. Similarly, PCR amplification was performed with primers adhE-F2 and adhE-R2 (specific sequences are shown in Table 2) to obtain the downstream homology arm fragment J, about 470 bp.

[0103] In the fourth step, the pV4 plasmid backbone fragment A, adhE-N20-gRNA sequence fragment H, and the upstream and downstream homologous arm fragments I and J of the adhE gene were assembled using the Golden Gate technology strategy (sequentially seamlessly connected in the order of A, H, I and J), and transformed into the competent cell TransT1 (Beijing Quanshijin Biotechnology Co., Ltd.). The obtained clone was verified by PCR using primers P15A-UP and adhE-R2, and the band size was about 1.3Kb. The plasmid DNA extracted from the positive clone was sent for sequencing analysis to obtain the correct pV4-del-adhE plasmid.

[0104] (4) The specific steps of adhE gene knockout are as follows:

[0105] Starting from the Escherichia coli engineered bacteria with the frd gene knocked out in step (2), plasmids pRedCas9 and pV4-del-adhE were simultaneously transformed into the electroporation competent cells with the frd gene knocked out in step (2), coated with kanamycin and chloramphenicol double-resistance plates, and placed at 30°C for overnight culture. Pick a single clone and place it in 2mL LB (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose), 250r / min speed, 30°C overnight to induce homologous recombination and cut DNA that has not undergone recombination. Dilute and apply LB plates (containing kanamycin and chloramphenicol, 2.5% L(+)-arabinose), and culture at 30°C overnight. Pick a single clone for colony PCR verification, primers are adhE-YZ-up and adhE-YZ-down, and the positive clones are amplified to obtain PCR products of about 872bp in size, and the positive clones are named recombinant Escherichia coli QJL005.

[0106] 4. Metabolic evolution to obtain Escherichia coli QJL008 and QJL010

[0107] Starting from QJL005, cell growth and lactate production capacity were simultaneously improved through evolutionary metabolism.

[0108] The fermentation medium used in Evolutionary Metabolism consists of the following components (the solvent is water):

[0109] Macroelements: glucose 50g / L, xylose 50g / L, NH4H2PO4 17.56g / L, (NH4)2HPO4 52.6g / L, MgSO4·7H2O 37g / L, betaine-HCl 15.361g / L, KCl 18.75g / L.

[0110] Trace elements: FeCl3·6H2O 2.4g / L, CoCl2·6H2O 0.3g / L, CuCl2·2H2O 0.15g / L, ZnCl2 0.3g / L, Na2MoO4·2H2O 0.3g / L, MnCl2·4H2O 0.5g / L, H3BO3 0.072g / L.

[0111] The evolutionary metabolic process used a 500 mL fermenter with 250 mL of fermentation medium and 4 M NaOH as a neutralizer to control the pH of the fermenter at 7.0.

[0112] In the 1st to 20th generations, the glucose concentration in the fermentation medium was 50 g / L (i.e., 5%); after acclimation for 34 days (transfer 20 times), the fermentation broth was transferred to a new fermenter to make the initial OD 550nm The obtained strains can be used for the next experiment, and one of the obtained strains is recorded as QJL008.

[0113] In the 20th to 40th generation, the glucose concentration in the fermentation medium was 50 g / L (i.e. 5%), and the xylose concentration was 50 g / L (i.e. 5%); after acclimation for 25 days (transfer 20 times), the fermentation broth was transferred to a new fermenter to make the initial OD 550nm Reached 0.05; after 40 generations of evolution in total, the obtained strain was recorded as QJL010.

[0114] Escherichia coli QJL010 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 12, 2023, with the deposit number CGMCC No.27325.

[0115] 5. Construction of strain QJL008-CRP*

[0116] Starting from Escherichia coli QJL008, the CRP G141D mutation was introduced using a two-step homologous recombination method to obtain recombinant Escherichia coli QJL008-CRP*. Using the DNA of the pXZ-CS plasmid (Tan et al., 2013 AEM 79: 4838-4844) as a template, PCR amplification was performed with primers crp-TB-cat-up / crp-TB-sacB-down to obtain a PCR product of about 3 kb, which is the first step homologous recombination fragment DNA fragment I, containing cat, sacB genes and 50 bp upper and lower homologous arm sequences.

[0117] In the second step, the pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97: 6640-6645; the plasmid was purchased from the Escherichia coli Collection Center of Yale University, USA) was first transformed into Escherichia coli QJL008 by electroporation, and then DNA fragment I was electroporated into Escherichia coli QJL008 carrying pKD46.

[0118] The electroporation conditions are as follows: first prepare electroporation competent cells of Escherichia coli QJL008 carrying the pKD46 plasmid (Dower et al., 1988, Nucleic Acids Res 16: 6127-6145); place 50 μl of competent cells on ice, add 50 ng of DNA fragment I, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV. After electroporation, quickly transfer 1 ml of LB culture medium to the electroporation cup, pipette 5 times, and then transfer to a test tube, incubate at 75 rpm and 30°C for 2 hours. Take 200 μl of bacterial solution and spread it on LB plate containing ampicillin (final concentration of 100 ug / ml) and chloramphenicol (final concentration of 34 μg / ml). After overnight culture at 37°C, select 5 single colonies for PCR verification using primers cat-up / crp-down (Table 2). Select a correct single colony and name it QJL008-CRP*C.

[0119] In the third step, the genomic DNA of QJL010 was used as a template and primers crp-YZ-up / crp-down were used to amplify the second homologous recombination DNA fragment II, which introduced the CRP G141D mutation and had a size of 919 bp.

[0120] In the fourth step, the second step homologous recombination DNA fragment II that introduced the CRP G141D mutation was electroporated into the strain QJL008-CRP*C.

[0121] The electroporation conditions were as follows: first, prepare electroporation competent cells of QJL008-CRP*C with pKD46 plasmid (see Dower et al., 1988 for preparation method); place 50 μl competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml LB culture medium to the electroporation cup, blow 5 times, transfer to a test tube, incubate at 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid medium without sodium chloride containing 10% sucrose (50 ml medium in a 250 ml flask), and after culturing for 24 hours, streak culture on LB solid medium without sodium chloride containing 6% sucrose. After sequencing verification, a correct single colony was selected and named QJL008-CRP* (Table 1). QJL008-CRP* has the following characteristics: the strain is obtained by replacing the CRP gene (SEQ ID No. 4) in QJL008 with the CRP G141D gene (SEQ ID No. 2).

[0122] Among them, the CRP G141D gene shown in SEQ ID No.2 encodes the CRP G141D protein shown in SEQ ID No.1, and the CRP gene shown in SEQ ID No.4 encodes the CRP protein shown in SEQ ID No.3.

[0123] Example 2: Fermentation evaluation of strains QJL008 and QJL008-CRP*

[0124] The seed culture medium consists of the following components (the solvent is water):

[0125] NaCl: 10g / L, Tryptone: 10g / L, yeast powder: 5g / L.

[0126] The fermentation medium consists of the following components (the solvent is water):

[0127] Macroelements: glucose 50g / L, xylose 50g / L, NH4H2PO4 17.56g / L, (NH4)2HPO4 52.6g / L, MgSO4·7H2O 37g / L, betaine-HCl 15.361g / L, KCl 18.75g / L;

[0128] Trace elements: FeCl3·6H2O 2.4g / L, CoCl2·6H2O 0.3g / L, CuCl2·2H2O 0.15g / L, ZnCl2 0.3g / L, Na2MoO4·2H2O 0.3g / L, MnCl2·4H2O 0.5g / L, H3BO3 0.072g / L.

[0129] Anaerobic fermentation was performed using Escherichia coli QJL008 and QJL008-CRP* as test strains, including the following steps:

[0130] (1) Seed culture: 30 mL of seed culture medium was placed in a 250 mL Erlenmeyer flask and sterilized at 115°C for 15 min. After cooling, single clones of recombinant Escherichia coli QJL008 and QJL008-CRP* were picked from the plate and inoculated into the seed culture medium. The culture was carried out at 37°C and 250 rpm for 12 hours to obtain seed solution, which was used to inoculate the fermentation medium.

[0131] (2) Fermentation culture: The volume of the fermentation medium in a 500 mL anaerobic tank was 250 mL, and the seed solution was added according to the final concentration OD 550nm =0.1 inoculation amount was inoculated into the fermentation medium, and fermented at 37°C, 150rpm, for 4 days to obtain a fermentation broth. The neutralizer was 6MKOH, and the pH of the fermentation tank was controlled at 7.0. The fermentation broth was all the substances in the fermentation tank. No gas was passed during the culture process.

[0132] Analysis method: Agilent-1200 high performance liquid chromatograph was used to measure the components in the fermentation broth after 96 hours. The concentrations of glucose, xylose and organic acids in the fermentation broth were determined using an Aminex HPX-87H organic acid analysis column from Biorad. The optical purity of lactic acid was analyzed using a SUMICHIRALO A-6000 chiral column from Sumika Chemical Analysis Service, Japan.

[0133] Glucose, xylose, lactic acid, formic acid, acetic acid, and succinic acid standards are all products of Merck Life Sciences, with catalog numbers 50-99-7, 58-86-6, 50-21-5, 64-18-6, 64-19-7, and 1190-53-0, respectively. The specific detection steps refer to the materials and methods of Zhu, XN; Tan, ZG; Xu, HT; Chen, J.; Tang, JL; Zhang, XL, Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli. Metab Eng 2014, 24, 87-96.). The results are shown in Table 4.

[0134] Table 4. Evaluation of anaerobic fermentation of recombinant Escherichia coli

[0135]

[0136]

[0137] The results showed that overexpression of the cAMP-independent cyclic adenosine monophosphate receptor protein mutant CRP G141D could significantly enhance the rate and ability of Escherichia coli to metabolize xylose (P < 0.001 (***)). In the presence of glucose, the rate of xylose consumption by strain QJL008 within 24 h was increased from 0.41 g / gDCW·h to 0.81 g / gDCW·h (P < 0.001 (***)). At the same time, the biomass in 24 h was increased from 0.57 g / L to 0.82 g / L (P < 0.001 (***)).

[0138] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Protein, any one of the following A1)-A3): A1) the protein shown in SEQ ID No. 1; A2) a protein having the same function as the protein described in A1) with one or more amino acid residues substituted and / or deleted and / or added to the amino acid sequence except for position 141; A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in A1) or A2).

2. The biological material related to the protein according to claim 1, which is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that: B1) The nucleic acid molecule is any one of the following b1)-b3): b1) the DNA molecule shown in SEQ ID No. 2 in the sequence list; b2) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) and encodes the protein of claim 1; b3) A DNA molecule that hybridizes with the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes the protein of claim 1.

4. A recombinant Escherichia coli containing a gene encoding the protein of claim 1.

5. The recombinant Escherichia coli according to claim 4, characterized in that: The recombinant Escherichia coli does not contain the gene encoding the protein of SEQ ID No.

3.

6. A method for preparing the recombinant Escherichia coli according to claim 4 or 5, characterized in that: The method uses Escherichia coli as a recipient bacterium, and comprises: introducing the coding gene of the protein of claim 1 into the genome of the recipient bacterium, or replacing the coding gene of the protein of SEQ ID No. 3 in the recipient bacterium, to obtain recombinant Escherichia coli.

7. A method for improving the ability of Escherichia coli to utilize xylose in the presence of glucose, comprising introducing the gene encoding the protein of claim 1 into the genome of a recipient Escherichia coli, or replacing the gene encoding the protein of SEQ ID No. 3 in Escherichia coli, thereby improving the ability of Escherichia coli to utilize xylose in the presence of glucose.

8. Use of the protein according to claim 1 or the biomaterial according to claim 2 or 3 for improving the ability of Escherichia coli to utilize xylose in the presence of glucose.

9. Use of the protein according to claim 1 or the biomaterial according to claim 2 or 3 in the co-utilization of glucose and xylose by Escherichia coli.

10. Use of the recombinant Escherichia coli according to claim 4 or 5 in the co-utilization of glucose and xylose by Escherichia coli.

Images